R.S. Snyder, Compiler
The IML-2 Final Report can be downloaded in Adobe Acrobat format from the Marshall Technical Reports Server (MTRS), located at the NASA Marshall Space Flight Center in Huntsville, Alabama. The report is located within the 1997 abstract subsection, once there locate the IML-2 Final Report (it should be #14 on the list) and click the link to download the file (caution, the file is 22.3 MB in size).
The IML-2 Final Report was published without a write-up for Dr. Richard Giege and his Crystal Growth of a Thermophilic Aspartyl-tRNA Synthetase experiment. That write-up appears below.
Crystal Growth of a Thermophilic Aspartyl-tRNA Synthetase
Aminoacyl-tRNA synthetases are the enzymes that attach specifically the amino acids to transfer RNA, and thus are responsible for the correct expression of the genetic code (1). Microgravity crystal growth experiments, using the Thermus thermophilus aspartyl-tRNA synthetase (ttAspRS) expressed in Escherichia coli (2), were carried out in APCF vapor diffusion and dialysis reactors provided by Dornier (Germany). Earth-grown crystals of this protein (a dimer of 2 x 68000) were already obtained and its structure solved (3). The fundamental objectives of this study were to obtain crystals of this protein in microgravity and to determine its influence on crystal growth and quality.
Highlights:
Preflight Studies on ttAspRS
The ttAspRS used in these assays was pure at 95% and monodisperse, assessed by gel electrophoresis and dynamic light scattering. Crystallization conditions were found from an extensive sparse-matrix screen (Hampton Research, Ca) and six different conditions, giving the best results, were established, refined and used to set the microgravity experiments.
These conditions were tested on ground in hanging drop reactors (HD) and in dialysis reactors (DIA) for 10 days at 20 degrees centigrade. All the crystals obtained during these preflight experiments were of orthorhombic morphology and their sizes varied from 0.1 to 0.2 mm in the longest dimension.
Flight Experiments
Experiments were conducted in 6 HD and 3 DIA reactors. The initial concentration of ttAspRS was 20 mg/ml in all assays. The precipitating agents in the HD reactors were, in 25 mM Tris-HCl pH 7.2:
- 25% or 30% sodium formate,
- 25% or 35% ammonium sulfate,
and also 30% (w/v) PEG 1500 alone or 28% (w/v) PEG 400 in 50 mM sodium Hepes pH 7.5, 200 mM calcium chloride.
The conditions containing sodium formate and 35% ammonium sulfate were also tested in the DIA reactors.
Among HD reactors only one did not yield crystals. The other ones produced crystals but of not sufficient size to be subjected to X-ray diffraction analysis.
After the flight, reactors were disassembled and the volumes of the protein samples were measured. A 6-fold reduction relative to the initial volume instead of a 2-fold one, as expected, was observed. As a result, the equilibrated concentration of protein in each reactor favored precipitation instead of crystallization, as predicted from the phase diagram studies on earth. Interestingly, numerous small crystals were observed in the space reactors, where only amorphous precipitates appeared in the same reactors on the ground. All crystallines in the droplets were birefringent but very fragile and readily solubilized in low salt conditions.
One hanging droplet contained crystals of significant sizes ranging from 0.1 to 0.3 mm that appeared to be remnants of one or more large crystals. These particules resembled shattered fragments with undefined morphologies and disrupted edges. It may have been possible that the crystals in this droplet were destroyed during manipulation in the course of deactivation or processes thereafter. Normally, ttAspRS that is equilibrated against 20-30% sodium formate or 25-35% ammonium sulfate crystallizes in an orthorhombic shape. In the pursuit of isolating and cleaning the crystal fragments for X-ray, 40% ammonium sulfate solution was used to rinse the crystal fragments away from precipitating debris as done for crystals grown on earth. However, these crystalline particles were extremely sensitive and fragile and ultimately dissolved upon washing. As the result, X-ray analysis could not be done. This surprising change in solubility properties may suggest that the crystalline fragments have been a unique crystal that grew under the influence of very high ammonium sulfate concentration. We cannot determine if it is a gravity effect since this has never been observed in any previous crystallization experiment on ground.
In all dialysis reactors we observed precipitation having strong appearance of denaturation. When the samples were dispensed out of the containers for close observation no protein crystals were found. Activity assays for the viability of ttAspRS showed inactivation of the enzyme in all dialysis chambers. Evidently, the enzyme has denatured for unknown reasons, which have contributed towards its propensity to precipitate instead of, crystallize.
Conclusions and Perspectives
The most dramatic observation obtained from space was the presence of precipitation in all reactors. We have speculated that the unexpected volume decrease in the HD chambers have changed the equilibrium conditions drastically as compared to those on earth and have placed the entire crystallization sample in the precipitating region of the phase diagram. Interestingly, many small crystallines were observed in the midst of precipitation which implies that nucleation still occurred and that the remaining soluble solutions are still supersaturated. However, when the enzyme is set for crystallization under the same conditions on ground, heavy precipitation would be observed with no trace of crystal nucleation. But as mentioned previously, it is difficult to determine if this is or not a gravity effect. Moreover, we have performed aspartate tRNA aminoacylation activity assays on the precipitants that grew under these conditions and found in all cases that the protein had retained activity.
On the contrary, the precipitants observed in the dialysis chambers had no enzymatic activity, which suggested evidence of structural unstability. The equivalent ground experiment produced fairly large crystals, which implies that microgravity or manipulation events may have affected the equilibrium process.
Since crystal growth is sensitive to volume changes, special attention may be worth taking in regards to the ability of each reactor to sustain an expected volume. In light of this, the volume parameter may be more reliable in DIA than in HD reactors. Moreover it has appeared that in the majority of the IML-2 experiments performed to date, dialysis chambers have produced the most successful results. Care in preparation of crystallization chambers should also be taken such as cleaning procedures or other exercises that may risk chemical contamination, which may affect the viability of the studied protein.
Concluding, this IML-2 experiment was an excellent preparation for the flight on USML-2, which gave excellent results. It allowed to better set crystallization conditions and to make the best choice of the reactor type. This IML-2 experiment showed also the propensity of ttAspRS to crystallize from precipitates by an Ostwald ripening mechanism, a fact that could also be found, but under different solvent conditions, on Earth (4).
References
Last Updated December 16, 1997